**3. Development of an efficient method for** *Agrobacterium***-mediated transformation for sugarcane**

The genetic improvement of sugarcane cultivars has been achieved to increase sugar productivity with the cultivars created by conventional breeding. The sugarcane (*Saccharum officinarum*) cultivars contributed high sucrose production and the backcrosses of *S. officinarum* resulted in elite cultivars of *S. hybrid* with higher yield, improving ratooning ability and disease resistance. However, these improvise achievements would still enable the sugar industries to face current issues on climate changes as well as higher sugar demand due to human population growth. Sugarcane has a highly complex genome, low fertilization and tiny seed which make traditional breeding highly difficult and laborious to create new sugarcane cultivars. Recent progress on molecular techniques could be used in sugarcane varietal improvement as well as the combination between both traditional and molecular breeding. Beginning with development of cell and tissue culture of sugarcane that has emerged as a valuable tool for various research activity in sugarcane improvement and propagation, sugarcane biotechnology has been used to introduce new traits that would be very difficult to conduct with conventional methods or almost not possible. The first establishment of genetic transformation method in sugarcane by Bower and Birch [35] and followed by engineering of agronomic traits using the method into various sugarcane genotypes are the important steps to support the development of sugarcane biotechnology. In addition, consideration also has been given to the development of molecular marker technologies for sugarcane breeding and variety identification [36].

of amino acids of glutamine and glycine in the media for stimulating multiplication of healthy sugarcane shoot will be suitable for future use in micropropagation as well as genetic trans-

Biotechnology of Drought-Tolerant Sugarcane http://dx.doi.org/10.5772/intechopen.72436 147

Genetic transformation technology serves as a useful and practical tool to introduce particular traits for crop improvement. Several genetic transformation methods have been attempted for delivery and expression of transgenes in plants. First genetic transformation methods for sugarcane were developed by direct introduction of desired genes using electroporation [47], polyethylene glycol (PEG) treatment [48], and particle bombardment [49]. These methods were considered less efficient compared to the indirect genetic transformation using *Agrobacterium*-mediated transformation. Furthermore, the direct transformation systems have certain limitations such as requires specific equipment, needs skill labor and results in high copy number integration in the plant genome [50]. The multiple gene copies frequently cause multiple gene arrangement, co-suppression and gene silencing [51]. Although, *Agrobacterium*-mediated transformation is restricted applicable in dicotyledonous plants due to the strictness of the host range of this bacteria, recent research indicated that *Agrobacterium*-mediated transformation is also possible in monocotyledonous plants such as in rice [52], maize [53] and banana [54]. In maize, the *Agrobacterium*-mediated transformation has been used for routine transformation using standard binary vector system and average stable transformation efficiency was 5.5% [55]. The evidences of the *Agrobacterium*mediated transformation system were also reported in sugarcane using meristematic explants [56, 57]. The *Agrobacterium*-mediated transformation method offers several advantages such as technical simplicity, low copy number and minimal genome rearrangement. Although *Agrobacterium*-mediated method has been applied also to sugarcane, the lack of reproducible results has been an obstacle to establish effective transformation protocol for routine genetic manipulation in the plants. The cell is being traumatic due to *Agrobacterium* infection and poor survival rate thereafter. Oxidative burst, phenolization, and subsequent cell death are frequent phenomena after the *Agrobacterium* infection [58]. Development of the *Agrobacterium*-mediated transformation is necessary to have reproducible and efficient methods in sugarcane. This section demonstrates an efficient transformation system for sugarcane using explant *in vitro* shoot generated from apical shoot tips to minimize bacterial

Genetic transformation system has been developed for sugarcane with distinct agronomically important traits, transformation methods, explant and culture condition. However, the use of *Agrobacterium*-mediated transformation method that considered more efficient was limited applying in sugarcane [58, 59]. Moreover, embryogenic callus was mostly reported as the explant for the *Agrobacterium*-mediated transformation system, but the use of callus includes the somaclonal variation. Direct regeneration from explants without an intervening callus phase has several advantages for *Agrobacterium*-mediated transformation in sugarcane. The isolated axillary bud explants from 6-months old field grown sugarcane were infected with *Agrobacterium* harboring the T-DNA of binary vector and resulted in stable transgenic sugarcane. The results suggested that the method can be achieved to generate transgenic sugarcane in about 5 months with transformation efficiency as high as 50% [42]. However, this transformation system needs numerous axillary buds as explants that should be isolated from sugarcane stalk and it is very difficult to avoid bacterial contaminant in the tissue culture

formation method.

contamination as well as somaclonal variation.

Plant cell has a totipotency ability to regenerate and differentiate into whole plant that completed with leafs, stem and root. The totipotency ability has been used for plant multiplication or micropropagation by inducing meristematic plant tissue in the medium supplemented with plant growth regulator to produce somatic embryogenesis callus, which in turn regenerate into whole plants. In sugarcane, the somatic embryogenesis callus is produced by induction of meristematic leaf tissue on the MS (Murashige and Skoog) medium containing 2,4-dichlorophenoxyacetic acid (2,4 D), and the embryogenic callus is then regenerated into whole plant on the MS free hormone [37]. Thus, considerable effort has been expended to use this micropropagation technique for providing the demand of numerous sugarcane seed. However, the application of tissue culture through somatic embryogenesis induces somaclonal variation in sugarcane [38, 39] that causes variants of phenotype, although it will be reverted to original parental thereafter [38]. Moreover, the presence of somaclonal variation is frequently used to obtain new type of sugarcane cultivars such as resistance to Fiji disease and mildew [40] and resistance to eyespot disease [41]. Although there is wide diversity of the usage, the somatic embryogenesis has been widely used as a part in genetic transformation system for the improvement of sugarcane cultivars [35].

Micropropagation of sugarcane can be also performed by direct regeneration of both apical and axillary meristem buds. The regeneration from axillary buds reduces the somaclonal variation events and is routinely used for mass multiplication of sugarcane [42, 43]. However, axillary buds isolated from field grown sugarcane stalk were frequently contaminated with bacteria and should use unexpected strong sterilant such as mercury chloride (HgCl<sup>2</sup> ) before cultured on MS media. Thus, the proper concentration should be carefully selected since this HgCl<sup>2</sup> sterilant is extremely harmful. Alternatively, *in vitro* sugarcane shoot can be regenerated from meristematic shoot apical and multiplied on MS media. There are various methods to improve the multiplication sugarcane shoot *in vitro* using MS media. Using temporary immersion system, shoot formation for sugarcane micropropagation was improved [44]. The shoot induction and multiplication on MS containing higher benzylamino purine (BAP) hormone resulted in weak, tiny, and nonseparable shoots, whereas the lower concentration resulted in desirable quality, well grown, easily separable, and healthy plantlets [45]. The media composition is considered to play an important role for achieving maximum growth rates on sugarcane tissue culture. Amino acids mixtures such as glutamine, glycine, asparagine, cysteine, and casein hydrolysate are frequently used as source of organic nitrogen in culture media [46]. Recently, addition of the amino acids mixture to the MS media has been examined in shoot multiplication of sugarcane. Addition of 2 ppm of glycine did not induce shoot multiplication, and 100 ppm of glutamine gave induction of shoot regeneration, but shoot growth rate was low. However, addition of the combination of glycine and glutamine resulted in well growth and healthy sugarcane plantlets. These results indicate that inclusion of amino acids of glutamine and glycine in the media for stimulating multiplication of healthy sugarcane shoot will be suitable for future use in micropropagation as well as genetic transformation method.

sugarcane biotechnology has been used to introduce new traits that would be very difficult to conduct with conventional methods or almost not possible. The first establishment of genetic transformation method in sugarcane by Bower and Birch [35] and followed by engineering of agronomic traits using the method into various sugarcane genotypes are the important steps to support the development of sugarcane biotechnology. In addition, consideration also has been given to the development of molecular marker technologies for sugarcane breeding and

Plant cell has a totipotency ability to regenerate and differentiate into whole plant that completed with leafs, stem and root. The totipotency ability has been used for plant multiplication or micropropagation by inducing meristematic plant tissue in the medium supplemented with plant growth regulator to produce somatic embryogenesis callus, which in turn regenerate into whole plants. In sugarcane, the somatic embryogenesis callus is produced by induction of meristematic leaf tissue on the MS (Murashige and Skoog) medium containing 2,4-dichlorophenoxyacetic acid (2,4 D), and the embryogenic callus is then regenerated into whole plant on the MS free hormone [37]. Thus, considerable effort has been expended to use this micropropagation technique for providing the demand of numerous sugarcane seed. However, the application of tissue culture through somatic embryogenesis induces somaclonal variation in sugarcane [38, 39] that causes variants of phenotype, although it will be reverted to original parental thereafter [38]. Moreover, the presence of somaclonal variation is frequently used to obtain new type of sugarcane cultivars such as resistance to Fiji disease and mildew [40] and resistance to eyespot disease [41]. Although there is wide diversity of the usage, the somatic embryogenesis has been widely used as a part in genetic transformation

Micropropagation of sugarcane can be also performed by direct regeneration of both apical and axillary meristem buds. The regeneration from axillary buds reduces the somaclonal variation events and is routinely used for mass multiplication of sugarcane [42, 43]. However, axillary buds isolated from field grown sugarcane stalk were frequently contaminated with

cultured on MS media. Thus, the proper concentration should be carefully selected since this

 sterilant is extremely harmful. Alternatively, *in vitro* sugarcane shoot can be regenerated from meristematic shoot apical and multiplied on MS media. There are various methods to improve the multiplication sugarcane shoot *in vitro* using MS media. Using temporary immersion system, shoot formation for sugarcane micropropagation was improved [44]. The shoot induction and multiplication on MS containing higher benzylamino purine (BAP) hormone resulted in weak, tiny, and nonseparable shoots, whereas the lower concentration resulted in desirable quality, well grown, easily separable, and healthy plantlets [45]. The media composition is considered to play an important role for achieving maximum growth rates on sugarcane tissue culture. Amino acids mixtures such as glutamine, glycine, asparagine, cysteine, and casein hydrolysate are frequently used as source of organic nitrogen in culture media [46]. Recently, addition of the amino acids mixture to the MS media has been examined in shoot multiplication of sugarcane. Addition of 2 ppm of glycine did not induce shoot multiplication, and 100 ppm of glutamine gave induction of shoot regeneration, but shoot growth rate was low. However, addition of the combination of glycine and glutamine resulted in well growth and healthy sugarcane plantlets. These results indicate that inclusion

) before

bacteria and should use unexpected strong sterilant such as mercury chloride (HgCl<sup>2</sup>

variety identification [36].

146 Sugarcane - Technology and Research

HgCl<sup>2</sup>

system for the improvement of sugarcane cultivars [35].

Genetic transformation technology serves as a useful and practical tool to introduce particular traits for crop improvement. Several genetic transformation methods have been attempted for delivery and expression of transgenes in plants. First genetic transformation methods for sugarcane were developed by direct introduction of desired genes using electroporation [47], polyethylene glycol (PEG) treatment [48], and particle bombardment [49]. These methods were considered less efficient compared to the indirect genetic transformation using *Agrobacterium*-mediated transformation. Furthermore, the direct transformation systems have certain limitations such as requires specific equipment, needs skill labor and results in high copy number integration in the plant genome [50]. The multiple gene copies frequently cause multiple gene arrangement, co-suppression and gene silencing [51]. Although, *Agrobacterium*-mediated transformation is restricted applicable in dicotyledonous plants due to the strictness of the host range of this bacteria, recent research indicated that *Agrobacterium*-mediated transformation is also possible in monocotyledonous plants such as in rice [52], maize [53] and banana [54]. In maize, the *Agrobacterium*-mediated transformation has been used for routine transformation using standard binary vector system and average stable transformation efficiency was 5.5% [55]. The evidences of the *Agrobacterium*mediated transformation system were also reported in sugarcane using meristematic explants [56, 57]. The *Agrobacterium*-mediated transformation method offers several advantages such as technical simplicity, low copy number and minimal genome rearrangement. Although *Agrobacterium*-mediated method has been applied also to sugarcane, the lack of reproducible results has been an obstacle to establish effective transformation protocol for routine genetic manipulation in the plants. The cell is being traumatic due to *Agrobacterium* infection and poor survival rate thereafter. Oxidative burst, phenolization, and subsequent cell death are frequent phenomena after the *Agrobacterium* infection [58]. Development of the *Agrobacterium*-mediated transformation is necessary to have reproducible and efficient methods in sugarcane. This section demonstrates an efficient transformation system for sugarcane using explant *in vitro* shoot generated from apical shoot tips to minimize bacterial contamination as well as somaclonal variation.

Genetic transformation system has been developed for sugarcane with distinct agronomically important traits, transformation methods, explant and culture condition. However, the use of *Agrobacterium*-mediated transformation method that considered more efficient was limited applying in sugarcane [58, 59]. Moreover, embryogenic callus was mostly reported as the explant for the *Agrobacterium*-mediated transformation system, but the use of callus includes the somaclonal variation. Direct regeneration from explants without an intervening callus phase has several advantages for *Agrobacterium*-mediated transformation in sugarcane. The isolated axillary bud explants from 6-months old field grown sugarcane were infected with *Agrobacterium* harboring the T-DNA of binary vector and resulted in stable transgenic sugarcane. The results suggested that the method can be achieved to generate transgenic sugarcane in about 5 months with transformation efficiency as high as 50% [42]. However, this transformation system needs numerous axillary buds as explants that should be isolated from sugarcane stalk and it is very difficult to avoid bacterial contaminant in the tissue culture media. By regeneration of *in vitro* shoot using meristematic shoot apical, subsequent multiplication in appropriate MS media will be suitable to overcome the problem of contaminant. The protocol for regeneration of *in vitro* shoot from shoot apical has been developed and with the method, the healthy shoot was rapidly multiplied in the MS media containing additional amino acid mixture of glutamine and glycine (**Figure 2A**–**F**). Green and healthy *in vitro* shoot from 2 to 4 weeks cultured was separated and basal segment that contains meristematic tissue was excised around 0.2–0.3 cm from the base (**Figure 2B** upper). These basal segments were injured with needles and used as the explant for *Agrobacterium*-mediated transformation. The injured or wounded tissue was suitable to induce *Agrobacterium* infection and allowed the *Agrobacterium* to penetrate into inner meristematic tissue of the basal segment. The presence of meristematic tissues provides young regenerable material that actively divided cell, competent for *Agrobacterium* infection, and improves the adhesion of *Agrobacterium* during co-cultivation [57]. Transient expression analysis showed that *Gus* gene expression was predominantly observed in the basal portion which was injured and contains meristematic tissue (**Figure 2B** lower). After cultured on selection medium containing the appropriate antibiotic for 2–3 weeks, the basal segment regenerates new axillar shoots, in which some of them become albino and bleached due to the presence of antibiotic in the media or regenerated green shoots. The explants with green shoots were transferred to the fresh selection medium and, after 5 times successive cycle, the putative transformant were acclimated. With this method, co-cultivation and antibiotic selection of putative transgenic shoot can be achieved in less than 4 months with transformation efficiencies around 6% when using 2 weeks-old shoot explant and the efficiencies sharply increased as high as 40% when using 4 weeks-old shoot explant. Genomic PCR and Southern Blot analysis indicated that most of the putative transformants contain insertion of the targeted DNA. All together these results suggest that basal segment of *in vitro* sugarcane shoot provides an effective explant for routinely *Agrobacterium*mediated transformation protocol and produces transgenic sugarcane.

The achievement of the current sugarcane transformation technology still needs further development. A number of undetermined conditions such as DNA promoter that drives the gene expression, selectable marker, *Agrobacterium* strain and some other factors are becoming important for improving genetic transformation efficiency. The promoter is a key DNA regulatory element that directs appropriate strength and pattern of gene expression in a constitutive or specific manner. Therefore, the promoter plays a crucial role in determining the transformation efficiency. There are some types of DNA promoters that drive strong, constitutive, or organ specificity expression. For example, the viral Cauliflower Mosaic Virus 35S (CaMV 35S) promoter has been widely used in the transformation of many dicot and monocot plants. However, it has been demonstrated that the expression activity of the 35S promoter was low in sugarcane [60]. The rice actin1 and Emu elements have shown to drive higher expression activity than CaMV 35S in different sugarcane tissues [61] and from the current research, it appeared that ubiquitin promoter has an emerging promoter for constitutive expression in sugarcane. The experiment concerning an effective promoter for sugarcane transformation has been also conducted using rice ubiquitin promoter (RUBQ2). The use of RUBQ2 promoter has increased transgene expression by about 1.6-fold over maize ubiquitin promoter in sugarcane [62]. Comparison study on GUS expression driven by CaMV35S and RUBQ2 promoter showed that RUBQ2 promoter produced high level GUS activity with clear blue spot in embryogenic callus and suspension cultures, while the CaMV35S promoter was not detected. Controversially, the GUS expression driven by sugarcane polyubiquitin promoter was dropped to very low or undetectable levels in the transgenic plants resulted from post-transcriptional gene silencing. Among the factors considered as limiting the recovery of transgenic plant is the involvement of selection marker in genetic transformation system. The selection of genetically transformed cell can be conducted through positive selection and negative selection. The positive selection is referred as those that promote the growth of transformed tissue and negative selection is the use selective agents, killing or fully inhibiting the growth of untransformed cell [63]. The use of gene for selectable marker in combination with targeted gene is directed to identify and allow surviving the transformed cell, and inhibit the growth of non-transformed cell in the media containing appropriate selective agents. Therefore, the use of selectable marker provides easy protocol to support proliferation of transformed cell and remove the un-transformed cell. Among the widely used selectable markers, the genes responsible for resistance of antibiotic kanamycin (*nptII*), hygromycin (*hptII*) and herbicide resistance (*bar*) are frequently applied to develop transgenic plants. The genes of *nptII*, *hptII* and *bar* inactivate the enzymes that play in role of antibiotic resistances neomycin phosphotransferase, hygromycin phosphotransferse and phosphinothricin acetyltransferase, respectively. Determination of the explant sensitivity to the antibiotic and the antibiotic concentration can be potentially effects of the successful genetic transformation. Exceeding high level of antibiotic is not only to kill the nontransformed cell, but also to give retardation for the growth of the transformed cell [64]. Evaluation of resistance to antibiotic kanamycin and hygromycin showed that the antibiotics can be used as selectable marker to obtain stable transformants in the cell suspension culture of the Gramineae such as *Triticum monococcum*, *Panicum maximum*, and *Saccharum officinarum* [65]. Comparison study on grapevine transformation revealed that both antibiotic kanamycin and hygromycin inhibited growth of the untransformed explant at 16 and 1 ug/mL, respectively. Due to the hygromycin which can be applied at lower level than kanamycin, hygromycin appears an appropriate

Biotechnology of Drought-Tolerant Sugarcane http://dx.doi.org/10.5772/intechopen.72436 149

**Figure 2.** Workflow of the *Agrobacterium*-mediated transformation using explant base segments of *in vitro* sugarcane. Green and healthy sugarcane *in vitro* shoot were micro-propagated in MS media and used as source of explants (A). Excised base segments of *in vitro* sugarcane were used as explant for the transformation (B upper) and clear blue spots of *Gus* gene expression were observed in the basal segments (B lower). Elimination of non-transformant and multiplication of putative transformant shoot in the selection MS media containing appropriate antibiotic (C and D). After five cycles in the selection media (E), the putative transformant were acclimated in greenhouse for further analysis (F). Arrow represents clear blue spots in the basal segment and albino shoot of non-transformant.

The achievement of the current sugarcane transformation technology still needs further development. A number of undetermined conditions such as DNA promoter that drives the gene expression, selectable marker, *Agrobacterium* strain and some other factors are becoming important for improving genetic transformation efficiency. The promoter is a key DNA regulatory element that directs appropriate strength and pattern of gene expression in a constitutive or specific manner. Therefore, the promoter plays a crucial role in determining the transformation efficiency. There are some types of DNA promoters that drive strong, constitutive, or organ specificity expression. For example, the viral Cauliflower Mosaic Virus 35S (CaMV 35S) promoter has been widely used in the transformation of many dicot and monocot plants. However, it has been demonstrated that the expression activity of the 35S promoter was low in sugarcane [60]. The rice actin1 and Emu elements have shown to drive higher expression activity than CaMV 35S in different sugarcane tissues [61] and from the current research, it appeared that ubiquitin promoter has an emerging promoter for constitutive expression in sugarcane. The experiment concerning an effective promoter for sugarcane transformation has been also conducted using rice ubiquitin promoter (RUBQ2). The use of RUBQ2 promoter has increased transgene expression by about 1.6-fold over maize ubiquitin promoter in sugarcane [62]. Comparison study on GUS expression driven by CaMV35S and RUBQ2 promoter showed that RUBQ2 promoter produced high level GUS activity with clear blue spot in embryogenic callus and suspension cultures, while the CaMV35S promoter was not detected. Controversially, the GUS expression driven by sugarcane polyubiquitin promoter was dropped to very low or undetectable levels in the transgenic plants resulted from post-transcriptional gene silencing.

media. By regeneration of *in vitro* shoot using meristematic shoot apical, subsequent multiplication in appropriate MS media will be suitable to overcome the problem of contaminant. The protocol for regeneration of *in vitro* shoot from shoot apical has been developed and with the method, the healthy shoot was rapidly multiplied in the MS media containing additional amino acid mixture of glutamine and glycine (**Figure 2A**–**F**). Green and healthy *in vitro* shoot from 2 to 4 weeks cultured was separated and basal segment that contains meristematic tissue was excised around 0.2–0.3 cm from the base (**Figure 2B** upper). These basal segments were injured with needles and used as the explant for *Agrobacterium*-mediated transformation. The injured or wounded tissue was suitable to induce *Agrobacterium* infection and allowed the *Agrobacterium* to penetrate into inner meristematic tissue of the basal segment. The presence of meristematic tissues provides young regenerable material that actively divided cell, competent for *Agrobacterium* infection, and improves the adhesion of *Agrobacterium* during co-cultivation [57]. Transient expression analysis showed that *Gus* gene expression was predominantly observed in the basal portion which was injured and contains meristematic tissue (**Figure 2B** lower). After cultured on selection medium containing the appropriate antibiotic for 2–3 weeks, the basal segment regenerates new axillar shoots, in which some of them become albino and bleached due to the presence of antibiotic in the media or regenerated green shoots. The explants with green shoots were transferred to the fresh selection medium and, after 5 times successive cycle, the putative transformant were acclimated. With this method, co-cultivation and antibiotic selection of putative transgenic shoot can be achieved in less than 4 months with transformation efficiencies around 6% when using 2 weeks-old shoot explant and the efficiencies sharply increased as high as 40% when using 4 weeks-old shoot explant. Genomic PCR and Southern Blot analysis indicated that most of the putative transformants contain insertion of the targeted DNA. All together these results suggest that basal segment of *in vitro* sugarcane shoot provides an effective explant for routinely *Agrobacterium*-

148 Sugarcane - Technology and Research

mediated transformation protocol and produces transgenic sugarcane.

**Figure 2.** Workflow of the *Agrobacterium*-mediated transformation using explant base segments of *in vitro* sugarcane. Green and healthy sugarcane *in vitro* shoot were micro-propagated in MS media and used as source of explants (A). Excised base segments of *in vitro* sugarcane were used as explant for the transformation (B upper) and clear blue spots of *Gus* gene expression were observed in the basal segments (B lower). Elimination of non-transformant and multiplication of putative transformant shoot in the selection MS media containing appropriate antibiotic (C and D). After five cycles in the selection media (E), the putative transformant were acclimated in greenhouse for further analysis

(F). Arrow represents clear blue spots in the basal segment and albino shoot of non-transformant.

Among the factors considered as limiting the recovery of transgenic plant is the involvement of selection marker in genetic transformation system. The selection of genetically transformed cell can be conducted through positive selection and negative selection. The positive selection is referred as those that promote the growth of transformed tissue and negative selection is the use selective agents, killing or fully inhibiting the growth of untransformed cell [63]. The use of gene for selectable marker in combination with targeted gene is directed to identify and allow surviving the transformed cell, and inhibit the growth of non-transformed cell in the media containing appropriate selective agents. Therefore, the use of selectable marker provides easy protocol to support proliferation of transformed cell and remove the un-transformed cell. Among the widely used selectable markers, the genes responsible for resistance of antibiotic kanamycin (*nptII*), hygromycin (*hptII*) and herbicide resistance (*bar*) are frequently applied to develop transgenic plants. The genes of *nptII*, *hptII* and *bar* inactivate the enzymes that play in role of antibiotic resistances neomycin phosphotransferase, hygromycin phosphotransferse and phosphinothricin acetyltransferase, respectively. Determination of the explant sensitivity to the antibiotic and the antibiotic concentration can be potentially effects of the successful genetic transformation. Exceeding high level of antibiotic is not only to kill the nontransformed cell, but also to give retardation for the growth of the transformed cell [64]. Evaluation of resistance to antibiotic kanamycin and hygromycin showed that the antibiotics can be used as selectable marker to obtain stable transformants in the cell suspension culture of the Gramineae such as *Triticum monococcum*, *Panicum maximum*, and *Saccharum officinarum* [65]. Comparison study on grapevine transformation revealed that both antibiotic kanamycin and hygromycin inhibited growth of the untransformed explant at 16 and 1 ug/mL, respectively. Due to the hygromycin which can be applied at lower level than kanamycin, hygromycin appears an appropriate selective agent [66]. Similar results reported that hygromycin is an effective selective marker for genetic transformation for monocot plants such as rice [67, 68], maize [69], and banana [70]. The herbicide Basta (*bar* gene) has been used as the selection marker for genetic transformation in rice [71], fescue—*Festuca arundinacea* [72], and oil palm [73]. However, limited reports have published the use of both *nptII*, *hptII*, and *bar* genes as effective selectable for sugarcane transformation. Using direct transformation with microprojectile bombardment, stable transformant was obtained after the selection of explant sugarcane callus on the media containing kanamycin and stepped increases in the antibiotic concentration allowed an active growing of callus, plantlets and completely inhibited untransformed callus [35]. When *Agrobacterium*mediated transformation was performed, similar results were observed using explant callus and *nptII* gene as the selectable marker. Regeneration transformant was successfully conducted by culturing the explant callus on the media containing 150 mg/L paromomycin sulfate [74]. However, the first successful report on *Agrobacterium*-mediated transformation for sugarcane used selectable marker *hptII* gene [57]. There are many reasons to elaborate the discrepancy between the effectiveness of selectable markers *nptII* or *hptII* genes, but hygromycin is much more effective than kanamycin for the selection of transformed cell and at low concentration, the hygromycin provides strong discrimination between transformed and nontransformed cell. Thus, hygromycin at the concentration of 25 mg/L is sufficient for routinely used *Agrobacterium*mediated transformation for sugarcane.

some plant species are accumulator of glycine betaine such as *Amaranthus*, sorghum, sugar beet and the other non-accumulators such as rice, sweet potato, and tobacco [5, 34]. Drought stress enhances accumulation of GB in the accumulator species for osmotic adjustment [82, 83]. Glycine betaine protects the plant cell by acting as an osmolyte, stabilizes protein and membrane cell, and maintains water balance during drought stress. It is widely accepted that the accumulation of GB plays an important role for the acclimatization of plant cell to drought stress. In many plants that do not accumulate GB, application of GB may help reduce adverse effects of the environment stress. The exogenous application of GB at 10 mM improved growth, leaf water content, and net photosynthesis, and increased growth and crop yield under environmental stress [84, 85]. However, consideration of economic and streamline useful application needs to be investigated. Determination of the GB concentration, timing, frequency of application, and a possibility of other disadvantage of exogenous GB application such as the risk of increasing pathogen attack should be well established [85]. Thus, genetic engineering for economically important crops such as rice, maize, and sugarcane that natu-

Biotechnology of Drought-Tolerant Sugarcane http://dx.doi.org/10.5772/intechopen.72436 151

Glycine betaine is an osmoprotectant found in wide range of microorganisms, plants, and animals that are synthesized under various environmental stresses [5]. Glycine betaine is mainly synthesized from choline as the substrate through two-step reactions, dehydrogenation of choline, and oxygenation of betaine aldehyde (**Figure 3**). In higher plants, choline is converted by choline monoxygenase (CMO) to betaine aldehyde, and then converted into glycine betaine by betaine aldehyde dehydrogenase (BADH) to GB [34, 86]. In microorganism and mammalian cells, GB is also synthesized by two-step pathway, but choline is converted to betaine aldehyde by choline dehydrogenase (CDH) and not by CMO, and then to GB by same BADH activity [34, 87]. In contrast, a single step-reaction catalyzed by choline oxidase (COD) for synthesis of GB is found in some microorganism such as *Arthrobacter globiformis* and *Arthrobacter panescens* [88] . In addition, a distinct substrate for GB synthesis is found in two halophytic microorganism *Actinopolyspora halophila* and *Ectothiorhodospira halochloris*. The GB is synthesized from substrate glycine by glycine methylation pathway [89]. The increasing knowledge of physiological pathway for GB biosynthesis as well as genomic engineering technology allow to create transgenic plants that are properly tolerant to drought stress by

The gene involved in the biochemical pathway can be used either to increase or diminish metabolite product by overexpressing or silencing the gene responsible for the metabolism. In the case of metabolite engineering of GB, the enzymes involved in the biochemical pathway have been focused as a potential target to engineer the content in the non-accumulator plants. For that reason, the genes encoding for the enzymes involved in pathway of GB biosynthesis have been cloned from various microorganisms and plants that accumulate GB. In microorganism, gene encoding CDH (*bet*A) and BADH (*bet*B) have been isolated from *Escherichia coli* [90, 91] and from salt-tolerant bacteria *Halomonas elongata* [92], whereas gene encoding COD or COX was cloned from soil-living bacteria *Arthrobacter panescens* and *Arthrobacter globiformis* [93, 94]. In higher plants, limited number of genes encoding CMO in combination with BADH has been cloned from spinach [95], sugar beet, and amaranth [96]. Genes responsible for GB synthesis from microorganism have become a major target in the genetic engineering of water stress-tolerant in plants that are unable to accumulate GB, such as tomato (*Solanum lycopersicum*), potato (*Solanum tuberosum*), rice (*Oryza sativa*),

rally are unable to accumulate GB will be an important target to improve.

engineering of glycine betaine biosynthesis.

*Agrobacterium* strain and density frequently have an impact on the plant genetic transformation efficiency. There are many *Agrobacterium* strains used for genetic transformation in plant and, among of them, the LBA4404 *Agrobacterium* strain is widely used for genetic transformation. The LBA4404 has a higher transformation efficiency in several plants such as in tobacco [75], wheat [76], and herb of *Bacopa monnieri* [77]. However, GV3101 *Agrobacterium* strain has been reported with highest transformation rate than EHA105, AGL1, and MP90 *Agrobacterium* strains in tomato [78]. In addition, concentration of *Agrobacterium* and wounding explants are also considered as the factors influencing transgene expression in loblolly pine [79]. Concentration of *Agrobacterium* at OD<sup>600</sup> = 0.5 improved the efficiency of transformation in cotton [80], whereas higher concentration will result in *Agrobacterium* overgrowth and difficulty to eliminate after co-cultivation. In sugarcane transformation system using explant *in vitro* shoot, GV3101 *Agrobacterium* concentration at 0.5 OD600 and wounding explants can be used for routinely transformation.
